Genetic Variation Among Cultivated Selections of Mamey Sapote (Pouteria Spp

Proc. Fla. State Hort. Soc. 117:195-200. 2004. GENETIC VARIATION AMONG CULTIVATED SELECTIONS OF MAMEY SAPOTE (POUTERIA SPP. [SAPOTACEAE])

SUSAN CARRARA1 ideas about the differentiation between the three closely re- Florida International University lated species which produce the fruit commonly called “ma- Department of Biological Sciences mey sapote” have the potential to enlarge the range of traits 11200 SW 8th Street that should be represented in such a collection. Miami, FL 33158 This paper aims to facilitate mamey sapote germplasm col- lection expansion and management by identifying geograph- RICHARD CAMPBELL ic areas of high genetic diversity for future collection. This is Fairchild Tropical Botanic Garden accomplished by analyzing the genetic diversity of selections 11935 Old Cutler Road held by FTBG and the University of Florida’s Tropical Re- Miami, FL 33156 search and Education Center (TREC) using the Amplified Fragment Length Polymorphism (AFLP) technique. RAYMOND SCHNELL The mamey sapote selections in this study can be grouped United States Department of Agriculture-Agriculture Research Service into three main categories based on the region in which they 13601 Old Cutler Road were collected: Cuba, the Caribbean coast of Central America Miami, FL 33158 (specifically the Yucatán Peninsula, the Petén of Guatemala, and Belize), and the Pacific coast of Central America (Guate- mala, El Salvador, Nicaragua, and Costa Rica at elevations Additional index words. Mamey sapote, Pouteria spp., genetic from sea level to 1000 m) (Fig. 1). Genetic diversity of selec- diversity, AFLP tions from the Yucatán peninsula were hypothesized to be low due to their morphological similarity and to the land clearing Abstract. Mamey sapote (Pouteria spp., Sapotaceae) is a tree that occurred there during the Spanish conquest only 500 fruit of economic and cultural importance in South Florida, Central America, Mexico, and the Caribbean. This study is years ago (Landa 1978). among the first to analyze genetic variability among cultivated Concurrently, selections from the Pacific coast popula- selections of mamey sapote. The Amplified Fragment Length tion were hypothesized to have greater variation because Polymorphism (AFLP) technique was used to estimate levels there are three species present in the region whose fruit is of genetic diversity in the germplasm collections of Fairchild considered mamey sapote. Pouteria sapota is currently cultivat- Tropical Botanic Garden and University of Florida. Although ed throughout Central America and the Caribbean in a range the collections overall represent a low level of genetic diversi- of soil types at altitudes up to 1,200 m. Pouteria viridis shares ty, higher levels of genetic diversity were found among selec- the same geographical range as P. sapota, but tends to grow at tions from Central America. This indicates future plant a higher altitude (1000-1500 m). Pouteria fossicola is distribut- collection in that region could capture greater genetic diversi- ed further south than P. viridis and P. sapota with a range ex- ty. This information can be applied to the management and ex- pansion of the germplasm collections by identifying duplicate tending from Nicaragua through Costa Rica to Panama selections, mislabeled plants, and locating geographical areas between 0 and 800 m above sea level (Pennington 1990). for future collection.

Knowledge of, and access to, the full range of a crop’s ge- netic resources is vitally important to the continuing develop- ment of agriculture. Genetic diversity, the total number of different alleles present in a species, is an important compo- nent of a plant’s genetic resources. The distribution and scale of a crop’s genetic diversity must be understood before effec- tive germplasm collection can be undertaken. Comprehen- sive genetic information allows curators of living collections to optimize the genetic diversity in their collections, plan for efficient collection enlargement, and ultimately furnish a wid- er selection of plants to growers. Mamey sapote is a well known crop in South Florida, and has been described by Balerdi et al. (1996) and Léon (2000). Fairchild Tropical Botanic Garden (FTBG) holds one of the most representative and genetically diverse collections in the USA. Yet, a greater range of morphological traits can be ob- served in mamey sapote’s native or cultivated range than exist in the FTBG germplasm collection. In addition, changing Fig. 1. Map indicating the Pacific and Caribbean regions from which ma- mey sapote selections in the germplasm collections of Fairchild Tropical Bo- 1Corresponding author. Present address: P.O. Box 16581, Asheville, NC tanic Garden (FTBG) and the University of Florida’s Tropical Research and 28816. Education Center (TREC) were collected.

Proc. Fla. State Hort. Soc. 117: 2004. 195

Fig. 2. UPGMA dendrogram of cultivated selections of mamey sapote in Fairchild Tropical Botanic Garden (FTBG) and the University of Florida’s Trop- ical Research and Education Center (TREC) germplasm collections in South Florida, USA.

196 Proc. Fla. State Hort. Soc. 117: 2004.

Table 1. Mamey sapote sample material used in AFLP analysis of genetic Table 2. Analysis of Molecular Variance (AMOVA) for cultivated selections diversity present in the Fairchild Tropical Botanic Garden (FTBG) and of mamey sapote in Fairchild Tropical Botanic Garden (FTBG) and the the University of Florida’s Tropical Research and Education Center University of Florida’s Tropical Research and Education Center (TREC) (TREC) germplasm collections in South Florida, USA. germplasm collections in South Florida, USA.

Population Group FTBG TREC Total Variance Source of variation df component Percentage Caribbean 13 1 14 Pacific 10 3 13 Caribbean versus Pacific regions Cuba 2 2 4 Between region 1 1.244 13% Florida 2 0 2 Within regions 27 8.216 87% Cuba or Florida 2 0 2 Control: same individuals distributed randomly into regions Other 2 0 2 Between regions 1 0.167 2% Unknown 0 6 6 Within regions 27 8.773 98% Total 31 12 43

Principal component analysis (PCA) supported the UPG- Materials and Methods MA-based cluster analysis. The first three axes summarized 20.6, 7.5 and 6.3% of the data set’s variability, respectively. A A total of 41 unique mamey sapote selections were analyzed, scatterplot of the first three principal components shows a including 29 individual selections from FTBG and 12 from tight cluster including the majority of the study selections, TREC (Table 1). Appendix 1 specifies collection location and with only the more diverse selections separating out (Fig. 3). replicate information for all selections included in this study. By removing outliers, the distribution can be seen more clear- Laboratory procedures were carried out in the USDA-ARS- ly (Fig. 4). SHRS Plant Sciences Laboratory using the ethanol-precipita- AMOVA results indicate 13% of the variance was attribut- tion based Epicentre MasterPure™ Plant Leaf DNA Purifica- able to genetic variation between the regions. This is higher tion Kit (Epicentre, Madison, Wis.). AFLP markers were than the between-region variance of control groups randomly generated using Applied Biosystem’s AFLP™ Ligation/Prese- assigned to populations (Table 2). Unfortunately, F statistics lective Amplification Module (Applied Biosystems, 2000). The are not relevant for use with AFLP data. protocol was modified by reducing reaction volumes by one Selection ‘2002-165 A’, which originated from a seed col- half to reduce costs and repeated twice for each selection. The lected in a fruit market in northern Costa Rica, is one of the resulting fragments were separated by capillary electrophore- more distinct selections. It was collected because the fruit had sis and fragment sizes were analyzed with Genotyper version morphological characteristics intermediate between P. sapota 3.7 (Applied Biosystems, Foster City, Calif.). and P. fossicola: a green skin with brown scruff on the nose. From the multitude of fragments produced, AFLP mark- Added to the morphological ambiguity between the two spe- ers were selected based on their consistency over two AFLP replicates of each sample. Fragment sizes identified by Geno- typer were manually verified. Mismatches between replicates were accepted as missing data, for a total of 1.4% missing data over a total of 104 markers. Pairwise similarity between samples was estimated using Nei and Li’s (1979) similarity coefficient, also known as the Dice coefficient, using NTSYS (Exeter Software, Setauket, N.Y.). Dendrograms were produced using the unweighted pair group method of analysis (UPGMA) (Sneath and Sokal, 1973). Confidence levels were placed on the dendrograms us- ing 2000 bootstrap replications with the program WinBoot (Yap and Nelson, 1996). Finally, principle component analysis (PCA) performed by SPSS (SPSS, Inc., Chicago, Ill.) was used as another visualization of the data. Analysis of Molecular Variance (AMOVA) was used to assess differentiation between the Pacific and Caribbean populations (Excoffier et al., 1992).

Results and Discussion

Some grouping of the selections according to their collec- tion location was observed in the UPGMA dendrogram but was not supported by the bootstrapping procedure, perhaps due to the high overall genetic similarity of the samples. How- ever, several trends can be noted. The majority of selections collected from the Yucatán and Cuba showed a high level of similarity. In addition, the selections collected in the Pacific Fig. 3. Principal component analysis of mamey sapote data based on AFLP region show greater dissimilarity from each other than do fingerprints of 65 cultivated selections of mamey sapote. The first three prin- cipal components account for 20.6, 7.5 and 6.3% of the data set’s variability, those from the Caribbean (Fig. 2). respectively.

Proc. Fla. State Hort. Soc. 117: 2004. 197

This study indicates that the range of genetic diversity present in the FTBG and TREC germplasm collections is dis- tributed unevenly. The majority of selections form a cluster with low overall genetic diversity, while a few selections in- crease the level of genetic diversity. Field observations of mamey sapote prior to this study have found greater morpho- logical variation throughout Central America than is current- ly present in the germplasm collection. In addition, AFLP analysis of those individuals in the germplasm collection with intermediate characteristics of P. sapota, P. viridis and/or P. fossicola indicate that these morphological variants repre- sent greater genetic variation. Inclusion of these variants would increase the genetic diversity represented in the germ- plasm collection.

Literature Cited

Applied Biosystems. 2000. AFLP™ Plant Mapping Protocol. Applied Biosys- tems, Foster City, CA. 58 pp. Balerdi, C. F., J. H. Crane, and C. W. Campbell. 1996. The Mamey Sapote. Horticultural Sciences Department Document FC-30. Florida Coopera- tive Extension Service, Institute of Food and Agricultural Sciences, Uni- versity of Florida. Available: http://edis.ifas.ufl.edu/MG331 Excoffier, L., P. E. Smouse, and J. M. Quattro. 1992. Analysis of molecular vari- ance inferred from metric distances among DNA haplotypes: application to human mitochondrial DNA restriction data. Genetics. 131:479-491. Fig. 4. Principal component analysis of mamey sapote data based on AFLP Landa, D. 1978. Yucatan before and after the conquest. Translated by fingerprints. Outliers have been removed. W. Gates. Dover Publications, Inc., New York. 162 pp. León, J. 2000. Botanica de los cultivos tropicales. Instituto Interamericano de Ciencias Agricolas. San José, Costa Rica. 522 pp. Nei, M. and W. Li. 1979. Mathematical model for studying genetic variation in terms of restriction endonucleases. PNAS USA. 76:5269-5273. cies, these results suggest that the traditional taxonomic dif- Pennington, T. D. 1990. Sapotaceae. Flora Neotropica Monograph 52. Orga- nization for Flora Neotropica, New York Botanic Garden. 770 pp. ferentiation between P. sapota and P. fossicola may need Sneath, P. H. and R. R. Sokal. 1973. Numerical taxonomy: the principles and reexamination. Because the selections from northern Costa practice of numerical taxonomy. W. H. Freeman and Company, San Fran- Rica and Nicaragua appear to be more dissimilar from each cisco, CA. 573 pp. other than do selections from any other location, future col- Standley, P. C. and L. O. Williams. 1967. Sapotaceae: Flora of Guatemala. Fieldiana: Botany 24:211-261. lecting in this region would increase the genetic diversity Yap, I. V. and R. J. Nelson. 1996. WinBoot: a program for performing boot- present in the FTBG collection more than would collecting strap analysis of binary data to determine the confidence limits of UPG- from Yucatán. MA-based dendrograms. IRRI Discussion Paper. Series No. 14, pp. 1-20.

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? Intermediate fruit characteristics Selected for rootstock (consistently high seed number). Hard exoderm, resistant to fruit flies, fruits in May Campeche Collected by Julian Lara Collected by Julian Lara Collected by Julian Lara ted from the Pacific or Caribbean regions. Collection location was not Nicaragua/Costa Rica location Population group Collection location Notes Germplasm Number of DNA extractions WhitmanGray Australia 1 1 FTBG FTBG Pacific Pacific known for all selections viridis viridis ‘Pace’‘Pantin’ 1‘Pantin’ 2‘Pantin’ 3‘Pantin’ 4‘Pantin’ 5‘Pantin’ 6‘Pantin’ 7‘Pantin’ 8‘Pantin’ 9‘Pantin’ 10 3 1 1 1 1 1 TREC FTBG 1 TREC 1 TREC 1 TREC 1 TREC 1 TREC Greenland Nursery Greenland Nursery Greenland Nursery Not applicable Greenland Nursery Not applicable Not applicable Not applicable Not applicable Not applicable Not applicable Cuba Not applicable Not applicable Cuba Not applicable Cuba Cuba Cuba Cuba Cuba Cuba Cuba Cuba S. Florida commercial cultivar S. Florida commercial cultivar S. Florida commercial cultivar S. Florida commercial cultivar S. Florida commercial cultivar S. Florida commercial cultivar S. Florida commercial cultivar S. Florida commercial cultivar S. Florida commercial cultivar S. Florida commercial cultivar Selection 2002-165 A 1 FTBG Pacific ‘9998’‘Alejos’‘AREC 3’‘Buena Vista’‘Cartagena’‘Casillo #1’‘Celso 2’‘Celso 3’‘Cepeda Especial’‘Chenox’‘Copan’ 1‘Danny’ 1 1 1‘Don Vicente’ 1‘Felipe Larguito’‘Felipe Mayo’ 1 1‘Florida’ TREC FTBG FTBG 1 TREC‘Gilberto’ 1Guatemala Seed FTBG‘Hawaii 1’ TREC 1 FTBG‘Jamaica’‘Lara’ 1 1 1 1 FTBG‘Lara’ 2 1 FTBG‘Lara’ 3 1 FTBG‘Lobo’ Caribbean Caribbean‘Lopez 1’ Not applicable 1 FTBG FTBG TREC 1‘Lopez 2’ Not applicable FTBG 1‘Lorito’ FTBG‘Magaña’ 1 Caribbean 1 Isle of Pines, Cuba‘Magaña’ 2 Caribbean TREC 1 Belize Campeche, Mexico Cartagena, Colombia‘Magaña’ 3 TREC Caribbean FTBG‘Marin’ 1 Selected for use as rootstock Deep red, fibrous fruit ‘Mayapan’ Caribbean 1 Red, sweet fruit, little fiber FTBG‘Morales’ Campeche, Mexico 1 Caribbean Caribbean Not applicableP. TREC Campeche, Mexico Caribbean 1 1P. Campeche, Mexico TREC Caribbean 1 Synonym of ‘Adelantado 2’ TREC Large fruit 1 Cuba Belize Seedling of ‘Alejos’ 1 TREC Caribbean 1 Seedling of ‘Alejos’ Campeche, Mexico Campeche, Mexico FTBG Pacific FTBG 1 Mexico or Belize FTBG Campeche, Mexico Not applicable 2 1 TREC Fruits in March (Campeche) FTBG TREC 1 Guatemala Came from Gary Zill FTBG Hawaii Orotina, Costa Rica TREC FTBG FTBG Pacific Not applicable Large fruit Not applicable Pacific Caribbean Pacific FL or Cuba Pacific FL or Cuba Jesus Maria, C. R. Not applicable Caribbean Campeche, Mexico Not applicable El Salvador Collected by Pedro Lopez El Salvador Small fruit and seed. Some green. Collected by Pedro Lopez Isle of Pines, Cuba Small fruit, strong red El Salvador Campeche, Mexico FL Large fruit Large fruit, red, little fiber Reputed scion of original Reputed scion of original Morales Collected by Dr. Appendix 1. List of mamey sapote selections analyzed with AFLP markers. Population group was only assigned to collec Appendix 1.

Proc. Fla. State Hort. Soc. 117: 2004. 199

Collected by Julian Lara Collected by Julian Lara Collected by Julian Lara Collected by Julian Lara Collected by Julian Lara ions collected from the Pacific or Caribbean regions. Collection loca- location Population group Collection location Notes Germplasm Number of DNA extractions tion was not known for all selections Selection ‘Pantin’ 11‘Pantin’ 12‘Pantin’ 13‘Pantin’14‘Pantin’ 15‘Pantin’ 16‘Pantin’ 17‘Pantin’ 18‘Pantin’ 19 1‘Pantin’ 20‘Piloto’ 1 1‘Piloto’ 2 1‘Piloto’ 3 1‘Piloto’ 4 1 Greenland Nursery‘Piloto’ 5 1 Greenland Nursery‘San Verde’ 1 Greenland Nursery‘Santo Domingo’ 1 Greenland Nursery Not applicable‘Tazumal’ 1 Greenland Nursery Not applicable‘Tichaná’ 3 Greenland Nursery Not applicable‘Tobias’ Greenland Nursery 1‘Vidal’ Not applicable Greenland Nursery Cuba 1 Not applicable‘Vidal Redondo’ Greenland Nursery Cuba 1 Not applicable‘Viejo’ FTBG Cuba 1 Not applicable 1 Cuba 1 1 Not applicable TREC Cuba Not applicable TREC Cuba TREC 1 Cuba TREC FTBG Cuba 1 TREC S. Florida commercial cultivar FTBG 1 Cuba 1 S. Florida commercial cultivar S. Florida commercial cultivar Not applicable 1 TREC S. Florida commercial cultivar FTBG S. Florida commercial cultivar FTBG 1 S. Florida commercial cultivar FTBG Cuba S. Florida commercial cultivar FTBG S. Florida commercial cultivar Pacific S. Florida commercial cultivar Pacific TREC Pacific Pacific Caribbean Pacific Omotepe, Nicaragua S. Florida commercial cultivar Jesus Maria, C. R. Caribbean Remnant tree, 200+ years Pacific El Salvador Campeche, Mexico Green skinned fruit Omotepe, Nicaragua Geronimo, Costa Rica Campeche, Mexico Round, red fruit 100+ years old Tree Giant, old remnant tree Possibly ‘Vidal’ El Salvador Appendix 1. (Continued)List of mamey sapote selections analyzed with AFLP markers. Population group was only assigned to select

200 Proc. Fla. State Hort. Soc. 117: 2004.